1. Field of the Invention
The present invention relates to a DNA-cutting enzyme, especially relates to a novel type II restriction endonuclease which recognizes and cuts DNA only at a particular sequence of nucleotides.
2. The Prior Arts
Restriction endonuclease is one of DNA-cutting enzymes found in bacteria. A restriction enzyme recognition sequence containing a two fold axis of symmetry. Therefore the recognition sequences from the 5′-end to the 3′-end are the same on either upper strand or lower strand of DNA duplex, and such symmetry is termed palindrome. For the nomenclature of restriction enzymes, the first 3 letters of the name refer to a strain of bacterium, which bacterium is the source of the enzyme, the following letter indicates the particular strain, and the last part of the name is a Roman numeral which indicates the order of discovery. For example, EcoRI was isolated from Escherichia coli (strain RY13).
Traditionally, the restriction endonucleases are divided into 3 groups, designated type I, type II and type III according to domain structure, cleavage position, specificity of recognition sequence, and cofactors requirements. Type I and type II enzymes are similar in that both have restriction endonuclease and methylase activities. Type I restriction enzymes bind to the recognition site and then cut randomly, somewhere thousand of bases from the recognition sequence. Type III enzymes cleave DNA around 24 to 26 bases along the length of the molecule. Type II restriction enzymes are found in late 1960's by Hamilton Smith et al., which recognize and cut DNA only at a particular sequence of nucleotides. Generally speaking, type II restriction enzymes recognize a specific sequence with 4 to 8 base pairs in length in double stranded deoxyribonucleic acid (DNA), and cleave specific site of the double helix DNA. Each restriction enzyme recognizes a specific sequence of nucleotide bases and cleaves the DNA along the molecule. Bacteria prevent their own DNA from being degraded by methylating their recognition sequences, which sequences are thus modified and protected from the endonucleases. The specific cleavage sites of restriction enzyme are close to the recognition sequences, and therefore restriction enzymes that recognize different nucleotide sequences can be purified from different bacterial species. They function like genetic scissors which allow DNA to be cut at desired sites and therefore become powerful tools in genetic or molecular manipulation.
The restriction enzyme and its corresponding methylase constitute the restriction-modification system (R-M) of a bacterial species. R-M system in bacteria protects against invasion of foreign DNA. The restriction endonuclease recognizes a specific sequence and the cognate methyltransferase modifies the same sequence to differentiate self-DNA from foreign DNA. Thousands of restriction enzymes have been purified and characterized.
Because of the abovementioned properties, the use of restriction enzymes are broadly applied in genetic engineering, DNA or gene cloning and gene mapping.
There are more than 20 putative R-M systems discovered in H. pylori 26695 and J99 strains based on sequence homology. Previous studies show that there are 14 Type II R-M systems with biochemical activities in H. pylori 26695 strain. The R-M systems of these two strains are very different when the complete sequences of 26695 and J99 strains are compared. The difference of R-M systems results in the barrier of interstrain plasmid DNA transfer and chromosomal DNA transformation. The biological significance of such diverse and complicated R-M systems in H. pylori is still unclear.
Isoschizomer are restriction enzymes that recognize the same sequence. However, the isoschizomers from different sources showed various sensitivities to different modified DNA. Therefore, several restriction enzymes from different sources but cutting the sequence at the same location will be employed together to obtain a better cleavage effect in the target nucleotide sequence.
The present invention therefore provides a restriction enzyme which can specifically recognize and cut a particular nucleotide sequence in order to provide alternative choices for cleaving DNA in the biotechnological manipulation of genetic engineering and gene cloning, and to improve the cutting efficiency. In addition, it offers a better cleavage effect for target nucleotide sequences which can not be efficiently cut with known restriction endonucleases.